Fat cells produce and secret the hormone leptin, the cytokine TNF-alpha, and the enzyme lipoprotein lipase, or LPL. All these proteins play key roles in the control of nutrition, energy balance, and insulin sensitivity as well as a variety of physiological conditions which are directly or indirectly connected with these parameters. Thus, secretion of physiologically important proteins, along with triglyceride storage and insulin-sensitive glucose transport, can be considered as one of the most important functions of adipose tissue. At present, it is not known whether secretion from adipose cells is constitutive or regulated, and membrane structures, such as secretory vesicles and/or granules which represent secretory pathway(s) in these cells have not yet been identified. We propose to explore the compartmentalization of leptin, TNF-alpha, LPL, and other secreted proteins in different subcellular fractions of rat adipocytes (endoplasmic reticulum, Golgi apparatus, plasma membrane, etc.) using either classical biochemical techniques (gradient centrifugations, agarose gel electrophoresis) or immunoadsorption with specific antibodies. We also propose to identify the constitutive secretory pathway in adipocytes and to compare intracellular compartmentalization of secreted molecules to constitutive secretory vesicles on one hand and to insulin-sensitive Glut4-containing vesicles, on the other. We will study the process of adipose cell secretion with the help of various secretagogues as well as specific inhibitors of key enzymes involved in membrane traffic, such as PI 3-kinase, Arf, V-type ATPase (proton pump) and others. In the process of this work, we expect to determine whether secretion and Glut4 translocation represent different pathways of membrane trafficking in adipocytes and to get an insight into the molecular mechanisms which control these pathways. In parallel, in collaboration with Dr.S.Farmer, we will analyze production and compartmentalization of the secreted molecules in cell lines which selectively express individual adipogenic transcription factors in order to determine the role of these factors in the onset of adipocyte s secretion. We will also determine whether or not the same genetic program is required for the onset of secretion and insulin-sensitive glucose uptake in differentiating adipose cells. Thus, we propose to provide compositional, functional, and developmental characterization of the secretory pathway(s) in adipocytes and to determine molecular differences as well as a possible cross-talk between this pathway and the insulin-regulated translocation of Glut4.